JPS6270757A - Method and apparatus for separation and analysis in liquid chromatography - Google Patents

Abstract

PURPOSE:To separate and purify a living body related substance and a water-sol uble macromolecular substance to analyze the same with high accuracy, by dialyzing the effluent from a chromatography column using a specific amphoteric ion exchange membrane. CONSTITUTION:An amphoteric ion exchange membrane is constituted of an anion exchange region and a cation exchange region or constituted of said two regions and a region having no ion exchange group. The membrane is formed so that when a 0.02mol/l aqueous KCl solution is contacted with one surface of the membrane and a 0.01mol/l aqueous KCl solution is contacted with the other surface of said membrane, the potential difference generated between both surfaces of the membrane is -8-+8mV and the membrane resistance in a 0.5mol/l aqueous KCl solution is 50OMEGA.cm<2> or less and the exchange capacity of a cation and an anion is 0.1-0.2mm equivalent/g. The effluent from a chromatography column is dialyzed by said amphoteric ion exchange membrane to remove the low MW electrolyte in the effluent. Therefore, protein and enzyme etc. can be rapidly separated and purified and the isolated substance can be analyzed with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method and apparatus for separating and analyzing by liquid chromatography, which separates and purifies a wide variety of substances quickly and with high efficiency. The present invention relates to a method and apparatus for separation and analysis using liquid chromatography, which can perform separation and analysis with high sensitivity.

(Prior art) In recent years, separation and analysis technology using liquid chromatography has rapidly advanced, and it has become possible to separate various substances from low to high molecular weight.However, in general, the eluent used in aqueous liquid chromatography (mobile phase) contains salts at a considerably higher concentration than the eluted sample, making it difficult to detect with an electrical conductivity meter, ultraviolet absorption meter, or differential refractometer when analyzing the eluted sample; However, when separating a sample into its respective components, there were problems such as desalting of the preparative sample.The desalting operation for such a sample was performed using a cellulose-based dialysis membrane. Diffusion dialysis using ultrafiltration membranes, ultrafiltration using ultrafiltration membranes, and electrodialysis can be considered, but in diffusion dialysis and ultrafiltration, the molecular
Samples with a value of 0 or less are not suitable for introduction into chromatography because they have a large membrane leakage and are not sufficiently desalted, and electrodialysis requires a complicated and large-sized device. Desalting to a salt concentration (less than several hundred ppm) is difficult. In addition, in separation by liquid chromatography, in order to stabilize the sample, it is necessary to introduce the electrolyte into the column solution from above and outside, such as by exchanging the column elution wave buffer. For similar reasons, it has been difficult to do so using conventional methods.

Furthermore, in liquid chromatography, particularly ion chromatography, attempts have been made to improve the sensitivity of analysis by treating column effluent with a membrane. (For example, Special Publication No. 58-66052, EP Publication No. 3277)
In this case, a cation exchange membrane is used between the column effluent and the regenerant, so cations can be exchanged, but other ionic impurities contained in the column effluent cannot be removed, resulting in a decrease in sensitivity. The improvement was not sufficient, and it was difficult to introduce the necessary snow melt into the column effluent.

(Problems to be Solved) The present invention solves the problems associated with aqueous liquid chromatography as described above, and provides a method for precisely separating and analyzing various substances with high sensitivity, simplicity, and high efficiency. The goal is to provide equipment.

(Another Means to Solve the Problem) The present invention provides a method for separation and analysis by liquid chromatography, and a device for treating zwitterionic ions by means of a liquid chromatography, which is characterized by performing dialysis treatment on liquid chromatography effluent using an amphoteric ion exchange membrane. The present invention relates to a separation/analysis device using liquid chromatography, characterized in that it is equipped with a dialysis device using an exchange membrane. However, the amphoteric ion exchange membrane referred to in the present invention is a membrane composed of a cation exchange region and an anion exchange region, or a membrane composed of these two regions and a region having no ion exchange group. The cation exchange region is a region having a functional group such as a carboxylic acid group or a sulfonic acid group that becomes negatively fixedly charged when ionized, while the anion exchange region is a region having a first, second, third or Fourth
This is a region that has a functional group that ionizes into a fixed positive charge, such as an ammonium base. These cation exchange regions and anion exchange regions are arranged alternately, and the distance between the centers of these adjacent regions is 1 or less, preferably 0.14 or less, and the exchange capacity of cations and anions is The amount is preferably 0.1 to 2 milliequivalents, preferably 0.3 to 2 milliequivalents, per dry 12, respectively. When an amphoteric ion exchange membrane that does not satisfy these conditions is used, the effects obtained by the present invention are significantly enhanced. In addition to the above, the following conditions are required for the amphoteric ion exchange membrane used in the present invention.

(When +to, oz mol/liter and 0.01 mol/liter potassium chloride aqueous solution are blocked by a membrane, the potential difference generated between both sides of the membrane is 18 to +8 mV, more preferably 14
(ii) the membrane resistance in an aqueous potassium chloride solution of o, s milliliter is 50 oh
If the amphoteric ion exchange membrane does not satisfy these conditions, the permeability of the membrane to the electrolyte will be low and the effect of the present invention will be diminished.

The amphoteric ion exchange membrane used in the present invention can be produced by various methods. For the manufacturing method, see "Membrane" Vol. 8, No. 4, pages 212-224, for example.
(1983). For example, a polymer poly A into which a cation exchange group can be introduced, a polymer poly B into which an anion exchange group can be introduced, and a polymer poly O into which an ion exchange group cannot be introduced as necessary.
After blending, a membrane with the desired shape is formed, anion and cation exchange groups are introduced, and if necessary, crosslinking or polymer polymer po l having cation exchange groups is performed.
Polymer poly that can introduce yA+ and anion exchange group
By introducing an anion exchange group into the film prepared by blending with B, it is possible to introduce a polymer poly A into which an ion exchange group can be introduced and a polymer pol having an anion exchange fund.
It is obtained by introducing cation exchange groups into the film produced by blending with y B-, and when blending these polymers, if necessary, further blending with polymer poly C that does not introduce ion exchange groups. Good too,
Or poly A% poly A+, poly B,
By introducing an ion exchange group into poly B- in advance,
Any of the following five block copolymers may be used. ) The obtained polymer may be subjected to blending, and in this case also crosslinking may be carried out if necessary. Another method is block copolymerization. For example, by block copolymerization, a polymer poly A into which a cation exchange group can be introduced, a polymer Fp o I yB into which an anion exchange group can be introduced, and a polymer poly O into which an ion exchange group cannot be introduced as necessary. A block copolymer in which poly A
A cation exchange group or an anion exchange group is introduced into polyH and polyH, respectively.

As an example of a block copolymer, (polyA-poly
B).

poly A-poly B-poly A, po
lyB-polyA-polyB.

polyA-polyo-polyB, polyA
-polyo-polyB-polyo, poly
o-polyA-polyo-polyB, poly
.

-poly A -poly O-poly B,
poly A-poly 0-polyB-poly
C-polyA, polyB-polyo-poly
Examples include linear bonds such as A-polyc-polyB, but there is a p
Preferably one containing olyc. Furthermore, a graft block copolymer may also be used. For example, polyB has polyA as a branch, polyA has polyB'ji branch
, (polyA-polyo)poly13 with n-type block copolymer on its branches, (polyB-poly
ly O) polyA with n-type block copolymer on its branches
.

polyA, polyA with polyo-poly B-polyo type block copolymer on its branches, polyo-polyA
-poly with polyo-type block copolymer as branches
B, po with poly A and polyLs as branches
After producing a graft block copolymer of various combinations of lyc'4m and forming it into a membrane, an amphoteric ion exchange membrane is obtained by introducing ion exchange groups into each,
In any of these cases, crosslinking may be performed depending on the purpose.

Here, the materials used to produce the amphoteric ion exchange membrane are: polymeric polyamide into which cation exchange groups can be introduced
Examples of A include styrene, α-methylstyrene, vinyltoluene, vinylnaphthalene, α-halogenated styrene, cyphenylfuchsien, o-, m-, p-chlorostyrene, o-, m-, p-hydroxystyrene. ,o
, m, p-hydroxystyrene derivatives (e.g. o, m,
Polymers of monomers having aromatic rings that can be easily sulfonated by known methods, such as p-methoxystyrene, o, m, p-acetoxystyrene (O+"+ p-tert-)) xystyrene, etc., or acrylics. Unsaturated carmenic acid esters such as acid esters, methacrylic acid esters, crotonic acid esters, conjugated diene carzene acid esters, monomers with cyano groups such as acrylonitrile, methacrylonitrile, vinylidene cyanide, or alkylidene malonic acid Esters or α-
Any polymer of monomers such as cyanoacrylates into which carmenic acid groups can be easily introduced by hydrolysis may be used. In addition, polymer polyB into which anion exchange groups can be introduced
For example, vinylpyridines, vinylpyrimidines, vinylquinolines, vinylcarbasoles, or (n is an integer from 1 to 3, R1 and FL2 each have 1 carbon number)
Any polymer of o, m, p-vinylphenylalkylene dialkylamines represented by the following alkyl groups (from 12 alkyl groups) can be used, and these polymers can be easily quaternized using an alkyl halogen compound. Further, the polymer polyo into which no ion exchange group is introduced may be a polymer of diene monomers such as butadiene, isoprene, pentadiene, cyclohexadiene, etc., and these can be easily crosslinked by known methods. In addition, in the process of producing an amphoteric ion exchange membrane, if sulfonation is not performed, polyo is styrene, vinyltoluene, α-
Aromatic compounds such as methylstyrene and vinylxibin
It may be a polymer of

In the blend or block copolymer described above, it is desirable that the polymer polyA into which a cation exchange group can be introduced and the polymer polyB into which an anion exchange group can be introduced are each contained in a weight ratio of 15 or more, and It is desirable that the molecular weight of each component polymer constituting these is 10 to 1021 moles.

In the liquid chromatography separation and analysis method of the present invention, a sample is placed into a chromatography column through an appropriate injection valve, and an eluent (mobile phase) is passed through the sample to completely or partially separate the sample chromatographically. The effluent from the column is sent to a flow path formed by an amphoteric ion exchange membrane. ultraviolet absorbance meter, differential refractometer, light scattering photometer, etc.)
The chromatogram peak is detected using the chromatogram peak, and the sample is finally collected for each section. However, in the channel formed by the amphoteric ion exchange membrane, the effluent from the column comes into contact with the 20,000 sides of the amphoteric ion exchange membrane. On the other side, the dialysate flows and the low molecular weight electrolyte in the column effluent is transferred to the dialysate side through the membrane, or the low molecular weight electrolyte from the dialysate side passes through the membrane to the column effluent side. However, the use of an amphoteric ion exchange membrane not only increases the rate of movement of low molecular weight electrolytes through the membrane, but also increases the resistance of electrolytes and non-electrolytes to permeate through the membrane. It is larger than conventional dialysis membranes, so ideal post-column processing can be performed with less sample loss. Furthermore, by using pure water or an aqueous solution containing an electrolyte with a lower molar concentration than the electrolyte contained in the column effluent as the dialysate, it is possible to completely remove the low molecular weight electrolytes in the column effluent. The accuracy of analysis can be improved. This effect is greater as the electrolyte concentration of the dialysate is lower. '-1, the dialysate contains glucose, saccharose, raffinose and other sugars, oligosaccharides,
It may contain about 10411 moles or more of a non-electrolyte substance such as alcohols, polyols, ethers, polyethers, ketones, acetonitrile, etc. at a concentration of 50 f/dt or less. Amphoteric ion exchange membranes have low permeability to non-electrolytes, and the osmotic pressure generated by these non-electrolytes has the effect of promoting the flow of electrolytes from the column effluent to the dialysate through the zwitterionic ion exchange membranes.
If the molecular weight is less than 1 mole, the amount of non-electrolyte flowing through the amphoteric ion exchange membrane into the column effluent cannot be ignored, interfering with chromatographic separation, purification or analysis, and reducing its sensitivity. descend.

When introducing a necessary low molecular weight electrolyte into the column effluent during dialysis treatment from the outside, it is sufficient to include the desired concentration of the electrolyte in the dialysate, and when replacing the electrolyte in the column effluent. The same is true for more efficient exchange, in which dialysis using an amphoteric ion exchange membrane is divided into two stages, and in the first stage of dialysis, low molecular weight electrolytes are extracted from the column effluent using the dialysate prepared as described above. After complete removal, this is achieved by introducing the electrolyte into the column effluent using a dialysate containing the necessary electrolyte in the second stage of dialysis.

The width of the channel formed by the amphoteric ion exchange membrane is increased in order to reduce the broadening of the sample band and to effectively remove or exchange electrolytes in the column effluent or introduce electrolytes into the column effluent. Narrow the membrane p in contact with the column effluent
It is necessary to increase the area. In the present invention, it is desirable that the cross-sectional area of the channel formed by the amphoteric ion exchange membrane is usually 0.07 to 20 mm, and its shape may be tubular, layered, rectangular, oval, triangular, or other. Any amorphous shape is acceptable. In addition, a part of the surface forming this flow path is made of a material other than the amphoteric ion exchange membrane, such as a polymeric material such as acrylic resin, polyethylene, polypropylene, polystyrene, fluorine resin, polyvinyl chloride, nylon, or stainless steel. It may be made of metal, glass, etc. Furthermore, if necessary, the channel formed by the amphoteric ion exchange membrane may be filled with an appropriate filler, which is particularly desirable when the cross-sectional area of the channel formed by the amphoteric ion exchange membrane is 2 or more layers. . Therefore, when the flow path formed by the amphoteric ion exchange membrane is tubular, the formation of spheres with a diameter of 02 to 0.8 times the inner diameter of the tube is a particle close to a spherical shape or If the flow path formed by the amphoteric ion exchange membrane has a shape other than a tubular shape, the diameter should be 02 to 08 times the maximum diameter of its cross section. The formation of spheres with is close to a spherical shape. On the other hand, since the electrolytes are mixed in the vertical direction, the membrane permeation rate of the electrolyte is increased, and (2) the flow path becomes narrow, so that the amplitude of the chromatogram is not increased.

In the present invention, the low molecular weight electrolytes that can migrate throughout the amphoteric ion exchange membrane are inorganic electrolytes and organic electrolytes with a molecular weight of 500 F1 mol or less. If the molecular weight exceeds 500 mol, it is unlikely that it will permeate the exchange membrane even if it is dissociated into ions. Furthermore, the molecular weight of the electrolyte for which better permeation can be expected is 2 oov 1 mol or less.

The concentration of electrolyte contained in the column effluent (eluent) is 2
It is desirable that the amount is mol/liter or less, and furthermore, 1
It is desirable that it be less than mol/liter. In addition, the column flow and effluent may contain agricultural organic non-electromagnetic substances at a concentration of 50 v/dt or less, but the column effluent may contain 5 SF/d.
When an organic non-Ti solution is contained in a concentration higher than L, the osmotic pressure difference may cause significant interference with electrolyte passage through the membrane. Therefore, in order not to impair the effects of the present invention, the dialysate should be converted into a molar concentration. It is desirable to include non-electrolytes at the same concentration or higher.

Next, the apparatus of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is a diagram showing one embodiment of the present invention, in which chromatography column C is packed with a granular packing material or gel, and samples introduced together with eluent from column population 2 are each collected here. The components are separated and flowed out from the column outlet 5. This eluent (mobile phase) is delivered from the eluent tank T to the liquid sending pump P1, via the conduit route l and, if necessary, to the line filter Ff, to the sample injection valve, together with the sample introduced from the sample injection port 3. Introduced into column C. The column effluent containing the sample separated into components is sent into the flow path of the dialysis device having a flow path formed using the amphoteric ion exchange membrane M, while the dialysate L2 is sent from the tank T2 to the pump P2. is sent from the inlet 6 of the dialysis device, and through the amphoteric ion exchange membrane, removes low molecular weight electrolytes contained in the effluent, exchanges them with electrolytes contained in the dialysate, or adds necessary electrolytes to the effluent. Electrolytes are introduced, etc. Thereafter, the effluent is sent to the detector S, and the dialysate is discharged from the outlet lower. A chromatogram is detected by a detector S, and each component is finally collected by a fraction collector FC. However, if the purpose is only for analysis, a final fraction collector is not necessary. The dialysis device using the amphoteric ion exchange membrane M is preferably one that incorporates, for example, a coiled tubular amphoteric ion exchange membrane.

In addition, if the electrolyte in the solution does not interfere with chromatogram detection and all necessary components are to be separated and purified, a liquid chromatography apparatus as shown in FIG. 2 is preferable. The difference between FIG. 2 and FIG. 1 is that the effluent from the chromatography column C is first sent to the detector S, a chromatogram is detected, and then 1, ? Only the effluent part containing the necessary components is sent to the dialysis machine by the valve ■, the low-molecular shielding electrolyte is removed, and the necessary components are collected by the fraction collector FC. be done.

Various packing materials for liquid chromatography columns are selected depending on the object to be separated, and examples include silica gel, octadecylsilane, octyl groups, phenyl groups, cyano groups, hydroxyl groups, amine groups, cation exchange groups, anion exchange groups, etc. There are porous polymers (gels) with chemically bonded silica gel cation exchange groups, anion exchange groups, and polar groups such as hydroxyl groups. Based on the separation mechanism using columns, liquid chromatography can be divided into ion pair chromatography, adsorption chromatography, affinity chromatography, partition chromatography, reversed phase chromatography,
It is classified into ion exchange chromatography, ion chromatography, gel permeation chromatography, etc.
Liquid chromatography in the present invention may be any of these methods.

[Example and comparative example]

Example 1 A pentablock compound in which polyisoprene (I), polybutadiene (B), polystyrene (S), and poly(4-vinylbenzyldimethylamine) (A) are bonded in the order of l-8-B-A-I. polymer (however, the weight percentages of the S part, A part, 1 part, and B part are each 27
%, 34%, 20% and 19%, and the overall number average molecular weight is 2. IX105? (1 mole) was dissolved in xylene to give 18 weight percent of polymer aI'fi. Next, this FG liquid is woven with polyethylene thread having a wire diameter of approximately 80 microns. It was applied to the tubular shape /A of a cod with an outside diameter of 1 mm (open area of about 5 degrees), and after slowly drying, it was treated in methyl iodide vapor to quaternize the A part and 20% by volume of chloride was added. The 8 moieties were fully crosslinked with I by treatment with a solution of sulfur in nitromethane, and the S moieties were sulfonated by further treatment with 2 volume percent chlorosulfonic acid in mW in dichloroethane. After washing with an acid aqueous solution and a saline solution, the cation and anion exchange capacities were measured by a conventional method and were found to be 0.80 meq. and 0.69 meq., respectively, per dry membrane IL?.

It is clear from this that an amphoteric ion exchange membrane was obtained. In addition, this amphoteric ion exchange membrane was immersed in an aqueous solution of IM lead acetate to stain the sulfonated S portion.
After cutting an ultra-thin section of size A, it was further treated with osmium tetroxide vapor and observed under a transmission electron microscope. As a result, a black layer (sulfonated S part, S), a white layer (crosslinked A lamellar structure was confirmed in which the B part, the striation 2, and the gray layer (quaternized A part, A+) were arranged in repeating units of -8--D-A+-D-.Thickness of each layer In addition, a 0.02 mol/liter KCt aqueous solution was poured into the inner cavity of this tubular amphoteric ion exchange membrane on the outer surface side above the OO 1 mol/liter KO6 aqueous solution. When the potential difference generated between the membrane surfaces of the amphoteric ion exchange membrane was measured using a conventional method using calomel electrode sound, it was +2.5 mV. (However, the outer surface side of the membrane was taken as a reference.) Using a silver electrode,
When the membrane resistance was measured in a 0.5M KC2 aqueous solution, it was found to be 4
．． It was 5 ohm-1.

The inner cavity of this tubular amphoteric ion exchange membrane is 0.5 m in diameter.
A Teflon thread was inserted and wound into a coil to create a dialysis device, and this was used to assemble the liquid chromatography device shown in Figure 2. Here, Column C was used by connecting two ft gel permeation chromatography columns G3000SW (inner diameter 75 fins, length 60 crn, Toyo Soda Kogyo Co., Ltd.) in series, and the detector was an ultraviolet absorbance meter. The chromatography conditions are as follows.

Eluent 0. IM phosphate buffer W (pH=7.0)1
0,2 M KCl (1 ml 1 minute) Dialysate Pure water sample Bovine serum albumin (r#% Fl 0.1 tonne) (injection volume, 0.5 mt) As a result, an aqueous solution containing the recovered albumin monomer During the separation, sinate ions, chloride ions, and potassium ions were all less than 1/100 of those in the eluent.At this time, the recovery rate of albumin was approximately 90%, and the chromatogram obtained using the dialysis device Peak broadening (increase in half-width) is
It was about 2C)%.

Further, when the recovered aqueous solution of albumin monomer was used as it was to carry out electrophoretic measurements using an agar gel in a conventional manner, the measurements could be carried out without any problems. As described above, it is clear that albumin can be easily separated and purified by using the method of the present invention.

Comparative Example 1 Bovine serum albumin was separated in the same manner as in Example 1 except that the dialysis device equipped with the amphoteric ion exchange membrane was not used. It was completely impossible to conduct electrophoretic measurements using the solution (7) as it was, since it contained the salt of (7).

Furthermore, when this aqueous solution was desalted using an ion exchange gel, only 40% of albumin could be recovered.

Example 2 In Example 1, the chromatography column was Phenyt-5PW (inner diameter 75, manufactured by Toyo Soda Kogyo Co., Ltd.).
The length was 7.5 cm), the detector used was an infrared absorbance meter and an ultraviolet absorbance meter in series, and the eluent was A (1.7M
Ammonium sulfate + 0.1 M phosphate buffer pH = 7.0) and B (0
, 1M phosphate buffer (pH = 7.0) to give a hernia gradient from A to B (60 minutes), and the other conditions were the same as in Example 1 to try to separate a mixture of cytochrome C, myoglobin, and zozyme. Ta. As a result, it was possible to detect the protein at an absorption wavelength other than that of water, and three completely separated chromatogram peaks were confirmed. Furthermore, when water was evaporated from the aqueous solutions containing each of the recovered proteins and the infrared absorption spectra were determined by I11, almost the same spectra as those for each pure protein were obtained.

Comparative Example 2 Proteins were separated in the same manner as in Example 2, except that the dialysis device equipped with the amphoteric ion exchange membrane was removed from the chromatography device.

When water was evaporated from the recovered aqueous solutions containing all of the proteins and infrared absorption spectra were measured, it was impossible to obtain spectra for the proteins contained because of interference with phosphate and ammonium sulfate.

Example 3 In Example 1, the chromatography column was replaced with IEX-210SC (manufactured by Toyo Shodatsu Kogyo Co., Ltd.) having a sulfonic acid group.
An attempt was made to separate a mixture of saccharose, glucose, and 7-lactose under the same conditions as in Example 1, except that the inner diameter was 7.5 m and the length was 60 cm, the detector was a differential refractometer, and the eluent was 004M saline. Ta.

As a result, these three types of sugars can be almost completely separated,
Both Na+ and Ct- ions contained in the recovered solution were 101) pm or less.

Implementation 114 In Example 1, the chromatography column was
ein A-5epharose CL-48 (Ph
armacia li'ine Chemicals)
Mouse serum was separated in the following manner using a column with an inner diameter of 7.5 and a fin length of 30 crn packed with f.

(1) Column with 0.05 M phosphate buffer (0.1
It was washed with 5M sodium chloride (pH=8.0) (OR).

(2) Mouse serum was diluted twice and equilibrated by dialysis with % C solution. It was sent from all sample loops at a rate of 0.2 mt/min to adsorb immune proteins onto the column.

(3) Liquid C was sent for cleaning.

(4) Solution C and 0.05 M < citrate buffer (0,15
A linear gradient was applied from C to D solution using M (containing common salt, pH=3.5) (D solution), and immunoglobulin (
Solutes of 1gG1, IgG2a + IgG2b' were performed.

At this time, the flow rate of the eluent was 0.5 ml/min, and the dialysate was placed in the dialysis machine equipped with the amphoteric ion exchange membrane at 0 and 0.
IM phosphate buffer (containing 0.15 M NaCl, pH
=7.2) and the flow rate was 3 mA/min.

As a result, immunoglobulin gG1. IgG2a, 1g
G25 has pH=6.0-7.0 and 4.5, respectively.
It was eluted in the range of ~5.0, 3.5 to 4.0, and the pH of these eluates was approximately 7, and the immunoglobulin could be kept stable even without special treatment such as dialysis. .

Comparative Example 3 In Example 3, the amphoteric ion exchange membrane was replaced with Experimental Bar 70 Lineate/Tube 811X (inner diameter 0.625, outer diameter 0,87
Separation of a mixture of saccharose, glucose, and fructose was attempted under the same conditions as in Example 3, except that the length was 21 nm). As a result, the saccharides were almost completely separated, but the recovered solution contained almost the same concentration of salt as the initial eluent.

Comparative Example 4 Saccharose and glucose were mixed under the same conditions as in Example 3, except that the amphoteric ion exchange membrane in Example 3 was changed to Hollow Fino® manufactured by Spectrum (fractionation molecules! 1000*force Kurogno 26132215. Number of fibers: 22). An attempt was made to separate a mixture of 7 lactoses. As a result, most of the sugars leaked from the membrane, and the recovery rate was less than 10%.

(Effects) As described above, the present invention is capable of treating saccharides, oligopeptides, amino acids, nucleic acid-related substances, antibiotics, coenzymes, vitamins, and organic acids by performing dialysis treatment on the chromatography column effluent using an amphoteric ion exchange membrane. , medium- and low-molecular-weight organic substances and proteins in various biologically related substances such as oligosaccharides and alcohols, natural water-soluble polymers such as enzymes, nucleic acids, and polysaccharides, or polyacrylic acid, polyethylene glycol, and polystyrene sulfone. The separation and purification of synthetic water-soluble polymers such as acids, polyamines, and polypeptides can be done quickly and efficiently, and liquid chromatography, which has traditionally not been used for qualitative analysis, can be performed using an amphoteric ion exchange membrane. The substance isolated by dialysis with N
It can be easily analyzed by MR method, infrared absorption spectroscopy, electrochemical measurement, electrophoresis, X pure scattering, elemental analysis, ultraviolet absorption spectroscopy, mass spectrometry, etc., so it can be widely used for qualitative analysis. It is particularly effective in analyzing biochemical substances, etc.

[Brief explanation of drawings]

1 and 2 illustrate an embodiment of the invention. Ll... Eluent; L2... Dialysate; L3... Extrusion liquid (usually pure water or the same aqueous solution as the dialysate): T1
... Eluent tank; T2 ... Dialysate tank; T6.
... Extrusion liquid tank; F... Line filter:
Pl, P2. P6...Liquid pump; B...Sample injection valve; C; Liquid chromatography column: D.
... Dialysis device equipped with an amphoteric ion exchange membrane; M... an amphoteric ion exchange membrane; S... a detector; R... a recorder: ■
...Flow path switching knob; Fc... Fraction collector: 1... Eluent conduit route; 2... Column population; 3... Sample inlet: 4... Sample overflow outlet ; 5... Column outlet; 6... Dialysate population; 7... Dialysate outlet; 8... Outlet outlet 1
Applicant Toyo Hakudatsu Kogyo Co., Ltd. Managing Director 1) Hiroshi Naka Figure 1 Figure 2 Procedural Amendments November 26, 1985 Commissioner of the Patent Office Michibe Uga 1, Indication of Case Patent Application No. 210074 of 1985 No. 2, Name of the invention Separation/analysis method and separation/analysis apparatus by liquid chromatography 1″ff1 3. Relationship with the case of the person making the amendment Patent applicant address 4560 Bold Tomita, Shinnanyo City, Yamaro Prefecture Name (3)
30) Satoshi Yamaguchi, Representative of Toyo-Senta Kogyo Co., Ltd.
Mei 4, Agent Address: 5th Floor, New Toranomon Building, 2-5-5 Toranomon, Minato-ku, Tokyo, Specification, Detailed Description of the Invention, Column 7-: ″″, -1 Corrected ``nilfdadiene'' to ``diphenylbutadiene.'' do. (2) On page 15, line L1, "olicosaccharide" is corrected to "oligosaccharide." (Here)

Claims (1)

[Scope of Claims] 1. A separation and analysis method using liquid chromatography, characterized in that the effluent from a chromatography column is subjected to dialysis treatment using an amphoteric ion exchange membrane. 2. The amphoteric ion exchange membrane is coated with a 0.02 mol/liter aqueous potassium chloride solution on one side of the membrane and 0.02 mol/liter on the other side.
When in contact with a 1 mol/liter potassium chloride aqueous solution, the potential difference generated between both sides of the membrane is -8 to +8 mV, and 0.
The method according to claim 1, wherein the membrane resistance in a 5 mol/liter potassium chloride aqueous solution is 50 ohm·cm^2 or less. 3. The method according to claim 1, wherein the cation and anion exchange capacity of the amphoteric ion exchange membrane is 0.1 to 2 milliequivalents per gram of dry membrane. 4. The method according to claims 1 to 3, wherein the amphoteric ion exchange membrane is a membrane consisting of a cation exchange region and an anion exchange region. 5. The method according to claims 1 to 3, wherein the amphoteric ion exchange membrane is a membrane consisting of a cation exchange region, an anion exchange region, and a region having no ion exchange group. 6. The method according to claims 1 to 5, wherein the amphoteric ion exchange membrane is a membrane in which cation exchange regions and anion exchange regions are arranged alternately. 7. The method according to any one of claims 1 to 6, wherein the distance between the centers of adjacent cation exchange regions and anion exchange regions of the amphoteric ion exchange membrane is 1 mm or less. 8. The method according to claim 1, wherein the dialysate used in the dialysis treatment is a non-electrolyte-free aqueous solution. 9. The method according to claim 1, wherein the dialysate used in the dialysis treatment is an aqueous solution containing 50 g/dl or less of non-electrolyte. 10. Claims 1 and 8, wherein the dialysis treatment is carried out by passing the column effluent through a tubular body composed of an amphoteric ion exchange membrane with an inner diameter of 0.3 to 5 mm.
, the method described in item 9. 11. The method according to claims 1 and 8 to 10, wherein the dialysis treatment is carried out by passing the column effluent through a tube made of a tubular amphoteric ion exchange membrane filled with a packing material. . 12. The method according to claim 11, wherein the filler is a spherical or nearly spherical particle or filament having a diameter of 0.2 to 0.8 times the inner diameter of the tubular amphoteric ion exchange membrane. 13. Liquid chromatography is one of ion pair chromatography, absorption chromatography, partition chromatography, affinity chromatography, reversed phase chromatography, ion exchange chromatography, ion chromatography, and gel permeation chromatography. A method according to claim 1. 14. A liquid chromatography separation/analysis device, characterized in that the liquid chromatography separation/analysis device is equipped with a dialysis device for chromatography effluent using an amphoteric ion exchange membrane. 15. The apparatus according to claim 14, wherein a dialysis device for chromatography effluent using an amphoteric ion exchange membrane is disposed immediately after the chromatography column. 16. The device according to claim 14, wherein a dialysis device for chromatography effluent using an amphoteric ion exchange membrane is disposed after the detection device. 17. The device according to claims 14 to 16, wherein the dialysis device incorporates a coiled tubular amphoteric ion exchange membrane.